Doctor of Philosophy, The Ohio State University, 2022, Microbiology

Leishmaniasis is a neglected protozoan disease affecting over 12 million people globally. Cutaneous leishmaniasis (CL) is the most common form, characterized by chronic skin lesions. Currently, there are no approved vaccines for human use. We have generated centrin knock out Leishmania (L.) mexicana (LmexCen-/-) mutants using CRISPR/Cas9. Centrin is a cytoskeletal protein required only for intracellular amastigote replication in Leishmania. Here, we investigated the safety, immunogenicity, and efficacy of LmexCen-/- parasites in vitro and in vivo. Our data shows that LmexCen-/- amastigotes present a growth defect, which results in significantly lower parasitic burdens and increased protective cytokine production in infected macrophages and dendritic cells, compared to LmexWT. Furthermore, LmexCen-/- parasites are safe in susceptible mouse models and efficacious against challenge with LmexWT in genetically different BALB/c and C57BL/6 mice. Vaccinated mice did not develop cutaneous lesions, displayed protective immunity, and showed significantly lower parasitic burdens compared to the controls. Overall, we demonstrate that LmexCen-/- parasites are a promising candidate vaccine against CL in pre-clinical models. Next, we explored the metabolic drivers of these vaccine-mediated immunological profiles. Metabolomics are emerging as a useful tool to uncover unknown networks that govern immune regulation and determine functional specialization. We analyzed the metabolic changes occurring after immunization with LmexCen-/- and compared them with LmexWT infection. Our results show enriched aspartate metabolism and pentose phosphate pathway (PPP) in ears immunized with LmexCen-/- parasites. These pathways are both known to promote M1 polarization in macrophages, and PPP in particular induces nitric oxide production in macrophages cultured with LmexCen-/-, suggesting a shift to a pro-inflammatory phenotype following immunization. Furthermore, immunized mice showed enriched t (open full item for complete abstract)

Committee: Abhay Satoskar (Advisor); Pravin Kaumaya (Committee Member); Steve Oghumu (Committee Member); Jesse Kwiek (Committee Member) Subjects: Immunology; Microbiology; Neurosciences; Parasitology

Doctor of Philosophy, The Ohio State University, 2023, Biomedical Sciences

Antibiotic resistance is an urgent public health problem and is associated each year with over a million deaths worldwide. Strategies to limit antibiotic exposures as well as improved practices for infection prevention and containment have been successful in reducing the emergence and spread of antibiotic resistance. Despite these efforts, novel therapeutic strategies are needed. In this dissertation, we investigate the induction of sugar-phosphate toxicities as a novel therapeutic modality to selectively target microbial pathogens. We use Salmonella enterica serovar Typhimurium, as our model pathogen, due to its clinical relevance, tractable genetics, and well-developed mouse models for studying infection. It is also one of the most characterized microbes and many aspects of its physiology and pathogenesis are relevant to other pathogens of the Enterobacteriaceae family for which antibiotic resistance is of paramount concern [i.e., the Carbapenem-resistant Enterobacteriaceae (CRE) and extended spectrum beta-lactamase (ESBL)-producing pathogens]. Here, we have assembled and characterized a collection of Salmonella mutants predicted to suffer sugar-phosphate toxicity due to the absence of targeted enzymes within a variety of sugar-utilization pathways. Elimination of these enzymes coupled with the provision of the appropriate sugar leads to the accumulation of toxic sugar-phosphate intermediates resulting in growth inhibition. These mutations serve as a proxy for small molecule inhibitors of those enzymes that would be used in real-world therapeutic applications. Of the seven mutants tested in vitro, five (galE, galT, rhaD, mtlD, and araD) mutants showed growth inhibition in addition to a fraB mutant reported in earlier work. All but the galT mutant were also attenuated in a mouse model of Salmonella-mediated gastroenteritis. While homologs of galE are widespread among bacteria and in humans, the araD, mtlD, rhaD, and fraB genes are rare in most phyla of bacteria a (open full item for complete abstract)

Committee: Brian Ahmer Ph.D. (Advisor); Venkat Gopalan Ph.D. (Committee Member); Vanessa Hale DVM, MAT, Ph.D. (Committee Member); John Gunn Ph.D (Committee Member) Subjects: Biomedical Research; Microbiology; Molecular Biology

Doctor of Philosophy (PhD), Wright State University, 2022, Biomedical Sciences PhD

Diffuse midline glioma, K27M-mutant (DMG) are intractable brain tumors, primarily occurring in the pediatric and adolescent population. Patients have a median survival of less than one year after diagnosis. A lack of therapeutic targets has been a barrier to improvement in patient survival. Irradiation therapy improves symptoms while chemotherapy and surgical intervention, for the most part, have not yet demonstrated utility for treatment. DMG are characterized by a histone H3 mutation that results in the genome-wide loss of epigenetic-repressive marks on K27 and is associated with the misexpression of genes, like Cancer/Testis antigens. A member of this group, transketolase-like 1 (TKTL1), is an enzyme that catalyzes reactions bridging glycolysis and the pentose phosphate pathway. This work demonstrates that DMG express TKTL1, which reprograms cellular metabolism in support of tumor cell growth and regulates the expression of hypoxia-inducible factor 1 alpha (HIF-1α). HIF-1α in tumor cells is associated with increased invasion, metastasis, increased survival, and resistance to therapy. Inhibition of HIF-1α in DMG cells decreased hypoxia-induced gene expression, glycolytic capacity, mitochondrial respiration, and tumor cell proliferation. TKTL1 knockdown similarly reduced tumor cell proliferation and the population of mitotic cells and decreased glycolytic rate. Loss of TKTL1 did not increase sensitivity to the chemotherapeutic agents, panobinostat or carboplatin, but rather slowed DMG cell growth independently of their cytotoxic effects. These data indicate TKTL1 expression in DMG alters metabolism and proliferation and is a potential therapeutic target.

Committee: Robert M. Lober M.D., Ph.D. (Committee Chair); Thomas L. Brown Ph.D. (Committee Co-Chair); Weiwen Long Ph.D. (Committee Member); David R. Ladle Ph.D. (Committee Member); Lynn K. Hartzler Ph.D. (Committee Member) Subjects: Biomedical Research; Cellular Biology; Oncology

Doctor of Philosophy, Case Western Reserve University, 2018, Biomedical Engineering

Adenosine Triphosphate (ATP) serves as the universal currency of energy in cellular systems. Hydrolysis of ATP thermodynamically drives the majority of cellular processes fundamental to life. The existence of a fast and robust method to observe ATP and its reactions in vivo would have profound applications both in the clinical diagnosis of metabolic abnormalities, and in the evaluation of therapies. Phosphorus-31 (31P) spectroscopy is the only modality capable of non-invasive non-destructive in vivo detection of ATP and its reactions. However, 31P spectroscopy methods are often challenging to perform due to two reasons. First, the instruments have an inherently low sensitivity to the biological signal. Second, conventional 31P spectroscopy methods have emphasized mathematical tractability rather than optimal signal detection. Consequently, 31P spectroscopy methods require long experiment times, and this has precluded their use in many applications. In this thesis, a new acquisition paradigm, the Magnetic Resonance Fingerprinting (MRF) framework, was applied to 31P spectroscopy method design in order to shorten experiment times. By prioritizing signal detection over mathematical tractability, the methods designed in this thesis sought to overcome the limitations imposed by instrument sensitivity and shorten experiment times. Success in this goal may enable new applications. Three main projects are described in this thesis. First, the MRF framework based 31P-MRF method was used to obtain efficient and simultaneous quantification of T1 relaxation time and concentration of multiple metabolites. This method was tested in simulation and validated ex-vivo. Second, sensitivity to magnetization transfer (MT) effects between phosphocreatine (PCr) and ATP was added to the 31P-MRF method to measure the in vivo chemical exchange rate of creatine kinase enzyme. This new method, the CK-MRF method, was assessed in vivo rat hindlimb. Finally, the 31P-MRF method was (open full item for complete abstract)

Committee: Dominique Durand PhD (Committee Chair); Chris Flask PhD (Committee Member); Mark Griswold PhD (Committee Member); Charles Hoppel MD (Committee Member); Nicole Seiberlich PhD (Committee Member); Xin Yu ScD (Advisor) Subjects: Biomedical Engineering; Medical Imaging